Potential Therapeutic Vulnerabilities Research Articles

CNSC-02. GLUTAMATERGIC SYNAPTIC INPUT DRIVES BRAIN METASTASIS OF BREAST CANCER AND MELANOMA

Abstract The nervous system is implicated in pathology of both cranial and extracranial cancers. In glioblastoma, direct excitatory glutamatergic synapses between neurons and cancer cells were discovered. Importantly, these neuron-cancer contacts drive glioblastoma proliferation and invasion. However, such synapses have not yet been documented in other types of brain cancer. In the context of brain metastases, indirect, peri-synaptic contacts between breast cancer cells and neurons were observed, which promote brain metastatic growth through glutamatergic signaling via NMDA receptors on the neoplastic cells. Recently, two studies have demonstrated that neuronal activity supports the growth of both the primary tumor and brain metastases from neuroendocrine carcinoma small cell lung cancer. In the work presented here, we demonstrate direct neuron-cancer synapses in early-stage brain metastases originating from breast cancer and melanoma. These synapses exhibited the typical structural features observed in synaptic connections and induced postsynaptic currents in the cancer cells through the activation of AMPA subtype glutamate receptors. Importantly, genetic and pharmacological perturbation of these receptors reduces the number of brain metastases and the overall metastatic burden in preclinical models of breast cancer and melanoma. These results uncover a previously unknown form of direct communication between neurons and brain-metastatic breast cancer and melanoma tumor cells. This neuronal-tumor synaptic interaction demonstrates the significance of the neuronal microenvironment in the early stages of the brain metastatic cascade. Furthermore, it unveils a potential therapeutic vulnerability associated with the neuronal microenvironment, suggesting that targeting the synaptic communication between neurons and cancer cells could be a promising strategy to impede the progression of brain metastases.

TP53 Deficiency in AML Confers Resistance to CAR T-Cells That Can be Overcome By Targeting the Cholesterol or Wnt Pathways

Introduction TP53-mutant acute myeloid leukemia (AML) and myelodysplastic neoplasms (MDS) are distinct clinicogenomic entities characterized by chemotherapy resistance and high relapse rates after allogeneic stem cell transplantation, and thus, represent an unmet clinical need. Chimeric antigen receptor (CAR) T-cell therapy might be a promising therapeutic option for TP53-mutant AML/MDS. However, the AML-intrinsic determinants of response to T-cell-based approaches in AML, in particular the impact of TP53-deficiency, are largely unknown. In the present study, we addressed the biologically and clinically highly relevant question as to whether, and, if so, how TP53 deficiency in AML cells might confer resistance to CAR T-cell therapy. Results We took advantage of recently developed, CRISPR/Cas9-engineered TP53 isogenic AML cell lines harboring TP53 null, missense, or wildtype alleles to exclude other potential genetic confounders of therapy resistance. Flow cytometry-based co-culture assays using these isogenic cell lines together with CAR T-cells targeting the common AML surface antigens CD33, CD371, CD123, and CD117, showed that AML cells harboring TP53 null or missense mutations were relatively resistant to CAR T-cells in vitro. CAR T-cells engaging TP53-deficient leukemia cells proliferated less, upregulated exhaustion markers, displayed increased trogocytosis, and were ultimately unable to control AML cell outgrowth. Live-cell imaging revealed a longer engagement time of CAR T-cells with TP53-deficient AML cells before killing occurred as judged by propidium iodide influx. Immunodeficient mice xenografted with TP53-deficient AML cells and treated with CAR T-cells exhibited significantly shortened survival compared to TP53-wildtype AML controls. Transcriptional profiling revealed that the cholesterol pathway was upregulated in TP53-deficient AML cells under CAR T-cell attack. Simultaneously, CAR T-cells engaging TP53-deficient AML demonstrated a downregulated Wnt pathway, in particular the master regulators of T-cell fate TCF7 and EOMES. Rational pharmacological targeting of either of these pathways, blocking by Simvastatin and activation by BIO-acetoxime, respectively, rescued TP53-deficient AML cell sensitivity to CAR T-cell-mediated killing in vitro. Discussion and Outlook Using a combination of in vitro co-incubation assays, live-cell imaging, gene expression profiling and a therapeutic in vivo model we could demonstrate that TP53 deficiency in AML cells confers relative resistance to CAR T-cell therapy and elucidate some of the underlying molecular mechanisms in leukemia target cells and CAR T-cells. We thus propose a model, in which the intrinsic apoptotic defect in TP53-deficient AML/MDS cells results in a longer duration of the cellular interaction between CAR T-cells and TP53-deficient AML/MDS cells. This longer temporal interaction eventually leads to reduced CAR T-cell proliferation and enhanced CAR T-cell exhaustion with an overall decrease in AML cell killing (figure 1). Furthermore, we identified the cholesterol pathway as a potential therapeutic vulnerability of TP53-deficient AML cells engaged by CAR T-cells, and the Wnt pathway as a promising avenue to enhance the efficacy of CAR T-cell therapy in TP53-deficient AML/MDS. Our data suggest that the combination of CAR T-cell therapies with pharmacological co-interventions - as exemplified in this study - may be a preferable strategy towards more efficacious and tolerable cellular therapies for patients with TP53-mutant myeloid neoplasms.

Proteomics on malignant pleural effusions reveals ERα loss in metastatic breast cancer associates with SGK1-NDRG1 deregulation.

Breast cancer (BCa) is a highly heterogeneous disease, with hormone receptor status being a key factor in patient prognostication and treatment decision-making. The majority of primary tumours are positive for oestrogen receptor alpha (ERα), which plays a key role in tumorigenesis and disease progression, and represents the major target for treatment of BCa. However, around one-third of patients with ERα-positive BCa relapse and progress into the metastatic stage, with 20% of metastatic cases characterised by loss of ERα expression after endocrine treatment, known as ERα-conversion. It remains unclear whether ERα-converted cancers are biologically similar to bona fide ERα-negative disease and which signalling cascades compensate for ERα loss and drive tumour progression. To better understand the biological changes that occur in metastatic BCa upon ERα loss, we performed (phospho)proteomics analysis of 47 malignant pleural effusions derived from 37 BCa patients, comparing ERα-positive, ERα-converted and ERα-negative cases. Our data revealed that the loss of ERα-dependency in this metastatic site leads to only a partial switch to an ERα-negative molecular phenotype, with preservation of a luminal-like proteomic landscape. Furthermore, we found evidence for decreased activity of several key kinases, including serum/glucocorticoid regulated kinase 1 (SGK1), in ERα-converted metastases. Loss of SGK1 substrate phosphorylation may compensate for the loss of ERα-dependency in advanced disease and exposes a potential therapeutic vulnerability that may be exploited in treating these patients.

Role of the Microenvironment in Glioma Pathogenesis.

Gliomas are a diverse group of primary central nervous system tumors that affect both children and adults. Recent studies have revealed a dynamic cross talk that occurs between glioma cells and components of their microenvironment, including neurons, astrocytes, immune cells, and the extracellular matrix. This cross talk regulates fundamental aspects of glioma development and growth. In this review, we discuss recent discoveries about the impact of these interactions on gliomas and highlight how tumor cells actively remodel their microenvironment to promote disease. These studies provide a better understanding of the interactions in the microenvironment that are important in gliomas, offer insight into the cross talk that occurs, and identify potential therapeutic vulnerabilities that can be utilized to improve clinical outcomes.

Regulation of SPDEF expression by DNA methylation in advanced prostate cancer.

Prostate cancer (PCa) presents a significant health challenge in men, with a substantial number of deaths attributed to metastatic castration resistant PCa (mCRPC). Moreover, African American men experience disproportionately high mortality rates due to PCa. This study delves into the pivotal role of SPDEF, a prostate specific Ets transcription factor, and its regulation by DNA methylation in the context of PCa progression. We performed Epigenetic reprogramming using daily treatment with non-toxic dose of 5Aza-2-deoxycytidine (5Aza-dC) for two weeks to assess its impact on PDEF expression in prostate cancer cells. Next, we conducted functional studies on reprogrammed cells, including cell migration (wound-healing assay), invasion (Boyden-Chamber test), and proliferation (MTT assay) to comprehensively evaluate the consequences of altered PDEF expression. We used bisulfite sequencing (BSP) to examine DNA methylation at SPDEF promoter. Simultaneously, we utilized siRNA-mediated targeting of key DNMTs (DNMT1, DNMT3A, and DNMT3B) to elucidate their specific role in regulating PDEF. We measured mRNA and protein expressions using qRT-PCR and immune-blotting techniques, respectively. In this report, we observed that: a) there is a gradual decrease in SPDEF expression with a concomitant increase in methylated CpG sites within the SPDEF gene during prostate cancer progression from lower to higher Gleason grade; b) Expression of DNMT's (DNMT1, 3a and 3b) is increased during prostate cancer progression, and there is an inverse correlation between SPDEF and DNMT expression; c) SPDEF levels are decreased in RC77/T, a line of PCa cells from African American origin similar to PC3 and DU145 cells (CRPC cells), as compared to LNCaP cells , a line of androgen dependent cells,; d) the 5' CpG island of SPDEF gene are hypermethylated in SPDEF-negative CRPC ( PC3, DU145 and RC77/T) cell lines but the same regions are hypomethylated in SPDEF-positive castrate sensitive (LNCaP) cell line ; (e) expression of SPDEF in PCa cells lacking SPDEF decreases cell migration and invasion, but has no significant effect on cell proliferation, and; (f) treatment with the demethylating agent, 5-aza-2'-deoxycytidine, or silencing of the DNMT's by siRNA, partially restores SPDEF expression in SPDEF-negative PCa cell lines, and decreases cell migration and invasion. These results indicate hypermethylation is a prevalent mechanism for decreasing SPDEF expression during prostate cancer progression. The data demonstrate that loss of SPDEF expression in prostate cancer cells, a critical step in cellular plasticity, results from a potentially reversible process of aberrant DNA methylation. These studies suggest DMNT activity as a potential therapeutic vulnerability that can be exploited for limiting cellular plasticity, tumor progression, and therapy resistance in prostate cancer.

2199P Immunohistochemical identification of clinical subtypes and potential therapeutic vulnerabilities of lung carcinoids based on multi-omic analysis

2199P Immunohistochemical identification of clinical subtypes and potential therapeutic vulnerabilities of lung carcinoids based on multi-omic analysis

Genetically Engineered Mouse Models of CIC-DUX4 Sarcoma Reveal an Unexpected Sensitivity to Histone Deacetylase (HDAC) Inhibition

Undifferentiated small round cell sarcomas constitute a highly aggressive subtype of soft tissue sarcoma that affects adolescents and young adults. Frequently, these cancers are caused by a gene fusion between the DNA binding domain of capicua (CIC), a canonical tumor suppressor that normally acts as a transcriptional repressor, with the transcriptional activation domain of double homeobox 4 (DUX4). Given the rare nature of CIC-DUX4 sarcoma (CDS), obtaining patient tissues to study the mechanisms of disease is very challenging. Moreover, conducting clinical trials to identify radiosensitizers or other novel treatments is not possible underscoring the need for a primary animal model. Here, we report the development and characterization of the first genetically engineered mouse model of CDS and the use of this model to investigate novel therapies. Using Cre-loxP technology, three genetically engineered mouse models of CDS were developed. The resulting tumors were characterized by hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and RNA-sequencing. Cell lines from multiple primary and metastatic tumors were derived and used for Chromatin Immunoprecipitation (ChiP) sequencing, Rapid Immunoprecipitation mass spectrometry of Endogenous proteins (RIME), and targeted drug screening. In all three models, spontaneous (Cre-independent) recombination occurred in chimeric animals resulting in the formation of soft tissue tumors, widespread metastasis to the lungs, liver, and brain, and 100% lethality within 4-8 weeks of life. The tumors histologically resembled human CDS and stained positive for an HA (hemagglutinin) epitope tag confirming transgene expression. Of 108 chimeric pups (generation 0, G0), only one male bred before succumbing to metastatic disease. G2 progeny now exhibit resistance to spontaneous recombination and tumor development enabling reliable propagation of one of the models for experimentation. Using tumor derived cell lines for integrative analyses of multi-omics datasets, we find evidence for a model where CIC-DUX4 functions as a neomorphic and oncogenic transcriptional activator, which cooperates with ETS family transcription factors to drive the CDS transcriptional program. Lastly, small molecule epigenetic drug screens in mouse and human CDS cell lines point to an unexpected sensitivity to HDAC inhibitors, which we hypothesize may be attributable to ETS factor hyperactivity. Genetically engineered mouse models of CIC-DUX4 sarcoma form tumors that recapitulate the human disease. Using mouse tumors and tumor-derived cell lines, we identify a mechanistic role and potential therapeutic vulnerability that converges on the ETS family transcription factors.

MDB-05. HUMAN FETAL CEREBELLAR CELL ATLAS INFORMS MEDULLOBLASTOMA ORIGIN AND ONCOGENESIS

Abstract Medulloblastoma (MB) is the most common malignant childhood brain tumor, yet the origin of the most aggressive subgroup-3 form remains elusive, impeding development of effective targeted treatments. Previous analyses of mouse cerebella or human counterparts from frozen tissue nuclei5 have not fully defined the compositional heterogeneity of MBs. Here, we undertook an unprecedented single-cell profiling of freshly-isolated human fetal cerebella to establish a reference map delineating hierarchical cellular states in MBs. We identified a unique transitional cerebellar progenitor connecting neural stem cells to neuronal lineages in developing fetal cerebella. Intersectional analysis revealed that the transitional progenitors were enriched in aggressive MB subgroups, including group-3 and metastatic tumors. Single-cell multi-omics revealed underlying regulatory networks in the transitional progenitor populations, including transcriptional determinants HNRNPH1 and SOX11, which are correlated with clinical prognosis in group-3 MBs. Genomic and Hi-C profiling identified de novo long-range chromatin-loops juxtaposing HNRNPH1/SOX11-targeted super-enhancers to cis-regulatory elements of MYC, an oncogenic driver for group-3 MBs. Targeting the transitional progenitor regulators inhibited MYC expression and MYC-driven group-3 MB growth. Our integrated single-cell atlases of human fetal cerebella and MBs reveal potential cell populations predisposed to transformation and regulatory circuitries underlying tumor cell states and oncogenesis, highlighting hitherto unrecognized transitional progenitor intermediates predictive of disease prognosis and potential therapeutic vulnerabilities.

Characterizing the landscape of RING-type E3 ubiquitin transferase-altered (RNF43 alt) colorectal cancer (CRC) and defining unique subsets with potential therapeutic vulnerabilities in microsatellite instability-high (MSI-H) CRC.

3134 Background: RNF43 is a tumor suppressor gene that represses the WNT/β-catenin pathway. Emerging data show that RNF43 alt are commonly present in MSI-H CRC with improved outcomes to immune checkpoint inhibitors (ICI). We evaluated whether genomic and transcriptomic analysis of MSI-H RNF43 alt CRC defines distinct subsets with potential therapeutic vulnerabilities. Methods: We analyzed CRC tumors (any stage/ subtype) sequenced with Tempus xT assay (DNA-seq of 648 genes at 500x coverage, full transcriptome RNA-seq). RNF43 alt were defined as pathogenic/likely pathogenic per OncoKB, or copy number aberrations (CNA); deep deletions, or amplifications (>8x). RNF43 alt vs. wildtype (wt) were compared using Chi-squared/Fisher’s Exact tests or Wilcoxon rank-sum tests to evaluate differences in biomarkers. Gene Set Variation Analysis (GSVA) was applied to gene expression data to derive single-sample pathway enrichment scores. ICI-treated MSI-H CRC from the MSKCC cohort (n = 74), publicly available in AACR GENIE v13, was used to assess the overall survival (OS) using the Kaplan-Meier method. Results: RNF43 alt was identified in 5.2% (635 of 12,243) CRC tumors. CNA accounted for 6% of RNF43 alt CRC. RNF43 alt vs. RNF43 wt CRC was associated with older age (median 69 vs. 59 years, p < 0.001), more prevalent in females (56% vs 43%; p < 0.001). A detailed comparison of RNF43 alt vs. RNF43 wt CRC with p < 0.001 are shown (Table). Of MSI-H CRC (n = 714), 51% were RNF43 alt, with 80% being RNF43 p.GLY659fs. Among MSI-H RNF43 alt CRC, BRAF alt was co-mutated in 59%, with BRAFV600E constituting 99%. Median TMB was higher for MSI-H RNF43 alt vs. MSI-H RNF43 wt (40 vs. 31 mut/MB, p < 0.001). Among MSI-H CRC, GSVA identified significant downregulation of Cetuximab_benefit pathway and upregulation of IGF1R and PI3K/AKT pathways in RNF43 alt vs RNF43 wt. Among ICI-treated MSI-H CRC (n = 74) from the MSKCC cohort, no significant OS difference (HR 0.81 [95%CI: 0.34 – 1.97]; p = 0.65) was noted for RNF43 alt (n = 42) vs. wt (n = 32). Conclusions: RNF43 alt are common in MSI-H CRC and associated with potentially actionable genomic and transcriptomic signatures. Functional characterization of interplay between the WNT signaling and therapeutically relevant pathways (PI3K/AKT, BRAF, IGF1R, Cetuximab benefit) to better define optimal ICI combination approaches in MSI-H RNF43 alt CRC is ongoing. [Table: see text]

Analysis of tumor- and circulating-free DNA methylation to identify clinically relevant small cell lung cancer subtypes.

8577 Background: Small-cell lung cancer (SCLC) is an aggressive malignancy composed of distinct transcriptional subtypes, defined by the predominant expression of one of the three transcription factors ASCL1 (SCLC-A), NEUROD1 (SCLC-N) and POU2F3 (SCLC-P) as well as an inflamed subtype (SCLC-I; see Gay et al. Cancer Cell. 2021), each with potential therapeutic vulnerabilities. Implementing subtyping in the clinic has remained challenging due to limited tissue availability, particularly for longitudinal monitoring. Given the known epigenetic regulation of critical SCLC transcriptional programs, we hypothesized that there would be subtype-specific patterns of DNA methylation that could be detected in tumor or blood from SCLC patients. Methods: We included 179 patients with SCLC and performed RNA sequencing and genomic-wide reduced-representation bisulfite sequencing (RRBS). We further analyzed DNA methylation in 68 plasma samples including longitudinal samples to track SCLC subtype evolution over time. Results: Using machine learning approaches, we developed a highly accurate DNA methylation-based classifier (SCLC-DMC) that could distinguish SCLC subtypes using clinical tumor samples with 95.8% accuracy in the testing set compared to mRNA-based profiling. We further adjusted the classifier for circulating-free DNA (cfDNA) to subtype SCLC from plasma. Using the cfDNA classifier (cfDMC), we could demonstrate that SCLC subtypes evolve frequently during disease progression, highlighting the need for longitudinal tracking of SCLC during clinical treatment. Furthermore, methylation-based subtyping predicted response to a wide variety of drugs in preclinical models like CDK and AURK inhibitors, and clinical outcomes were indistinguishable in cohorts of patients subtyped using mRNA or SCLC-DMC (p = 0.95). Conclusions: These data establish that tumor and cfDNA methylation can be used to identify SCLC subtypes and guide precision SCLC therapy.

Dual inhibition of CDK12 and CDK13 uncovers actionable vulnerabilities in patient-derived ovarian cancer organoids

BackgroundHigh grade serous ovarian cancer (HGSOC) is highly lethal, partly due to chemotherapy resistance and limited availability of targeted approaches. Cyclin dependent kinases 12 and 13 (CDK12/13) are promising therapeutic targets in human cancers, including HGSOC. Nevertheless, the effects of their inhibition in HGSOC and the potential synergy with other drugs are poorly known.MethodsWe analyzed the effects of the CDK12/13 inhibitor THZ531 in HGSOC cells and patient-derived organoids (PDOs). RNA sequencing and quantitative PCR analyses were performed to identify the genome-wide effects of short-term CDK12/13 inhibition on the transcriptome of HGSOC cells. Viability assays with HGSOC cells and PDOs were performed to assess the efficacy of THZ531 as single agent or in combination with clinically relevant drugs.ResultsThe CDK12 and CDK13 genes are deregulated in HGSOC and their concomitant up-regulation with the oncogene MYC predicts poor prognosis. HGSOC cells and PDOs display high sensitivity to CDK12/13 inhibition, which synergizes with drugs in clinical use for HGSOC. Transcriptome analyses revealed cancer-relevant genes whose expression is repressed by dual CDK12/13 inhibition through impaired splicing. Combined treatment with THZ531 and inhibitors of pathways regulated by these cancer relevant genes (EGFR, RPTOR, ATRIP) exerted synergic effects on HGSOC PDO viability.ConclusionsCDK12 and CDK13 represent valuable therapeutic targets for HGSOC. We uncovered a wide spectrum of CDK12/13 targets as potential therapeutic vulnerabilities for HGSOC. Moreover, our study indicates that CDK12/13 inhibition enhances the efficacy of approved drugs that are already in use for HGSOC or other human cancers.

A Non-Canonical Role for the Glycosyltransferase Enzyme UGT2B17 as a Novel Constituent of the B Cell Receptor Signalosome.

In chronic lymphocytic leukemia (CLL), an elevated glycosyltransferase UGT2B17 expression (UGT2B17HI) identifies a subgroup of patients with shorter survival and poor drug response. We uncovered a mechanism, possibly independent of its enzymatic function, characterized by an enhanced expression and signaling of the proximal effectors of the pro-survival B cell receptor (BCR) pathway and elevated Bruton tyrosine kinase (BTK) phosphorylation in B-CLL cells from UGT2B17HI patients. A prominent feature of B-CLL cells is the strong correlation of UGT2B17 expression with the adverse marker ZAP70 encoding a tyrosine kinase that promotes B-CLL cell survival. Their combined high expression levels in the treatment of naïve patients further defined a prognostic group with the highest risk of poor survival. In leukemic cells, UGT2B17 knockout and repression of ZAP70 reduced proliferation, suggesting that the function of UGT2B17 might involve ZAP70. Mechanistically, UGT2B17 interacted with several kinases of the BCR pathway, including ZAP70, SYK, and BTK, revealing a potential therapeutic vulnerability. The dual SYK and JAK/STAT6 inhibitor cerdulatinib most effectively compromised the proliferative advantage conferred by UGT2B17 compared to the selective BTK inhibitor ibrutinib. Findings point to an oncogenic role for UGT2B17 as a novel constituent of BCR signalosome also connected with microenvironmental signaling.

Abstract IA07: Molecular characterization of acquired resistance to KRASG12C-EGFR inhibition in colorectal cancer

Abstract Until recently, efforts to pharmacologically target KRAS have been unsuccessful due to its small binding pocket, high affinity for GTP, and redundant mechanisms of posttranslational processing. The development of allele specific KRAS G12C inhibitors that trap KRAS in the inactive, GDP-bound, state led to a paradigm change and clinical responses in 30% of non-small cell lung cancer (NSCLC) patients harboring KRAS G12C mutations. KRAS G12C inhibitors, such as sotorasib and adagrasib, have rapidly moved through clinical development and are poised to transform care of patients with KRAS G12C mutant cancers, in particular NSCLC. However, these agents are not as effective in colorectal cancers (CRC) with KRAS G12C mutation. We have previously shown that the activity of these drugs in KRAS G12C CRC is limited because activation of epidermal growth factor receptor (EGFR) reactivates ERK signaling therefore combinatorial KRAS G12C and EGFR inhibition more effectively targets KRAS G12C CRC. Early trial data provide clinical support for this observation as anti EGFR monoclonal antibodies combined with sotorasib or adagrasib double the overall response to single agents in KRAS G12C mutant CRC patients. Based on these data, these KRAS G12C and EGFR antibody combination treatments are being evaluated in registrational, phase 3 trials. Nonetheless, patients treated with these agents, alone or in combination, eventually acquire secondary resistance. Several studies have characterized resistance to KRAS G12C monotherapy, in this work, we sought to determine the mechanisms of acquired resistance to concomitant KRAS and EGFR inhibition in CRC. In cell lines, patient-derived xenograft, and patient samples, a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drug can be detected at progression. Serial analysis of patient blood samples during treatment demonstrates that most of these alterations are detected at a low frequency that does not increase substantially and sometimes disappears over time, with the exception of KRAS G12C amplification which rises in step with tumor marker levels and clinical progression. Here we show that a CRC cell line that acquired resistance to sotorasib-cetuximab combination through KRAS G12C amplification undergoes oncogene-induced senescence upon drug withdrawal. Senescence markers were detected in a tissue biopsy from a patient at relapse after treatment stop, and amplified KRAS G12C signal in circulating DNA from relapsed patients rapidly recedes after treatment interruption. These data indicate that while most resistance-associated alterations to KRAS-EGFR coinhibition remain subclonal, affecting a small population of cells, KRAS G12C amplification is a recurrent resistance mechanism that drives a higher portion of resistance over time and provides a potential therapeutic vulnerability, whereby therapies that target the senescence response at drug withdrawal may overcome resistance to KRAS G12C-EGFR inhibition. Citation Format: Sandra Misale. Molecular characterization of acquired resistance to KRASG12C-EGFR inhibition in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA07.

CBX2-mediated suppression of SIAH2 triggers WNK1 accumulations to promote glycolysis in hepatocellular carcinoma.

Previous studies have highlighted the poor prognosis of liver cancer, and treatment effects are overall limited. We aimed to confirm the biological roles of SIAH2 in liver cancer and provide potential therapeutic targets. Differential analysis was conducted based on public datasets and found that SIAH2 expressed lowly in HCC samples relative to normal tissues, which was demonstrated in tumor samples via immunohistochemistry (IHC). Besides, SIAH2 overexpression could significantly suppress HCC proliferation. SIAH2 deficiency induced cell proliferation, migration and self-renewal abilities in vitro and in vivo. Mechanistically, SIAH2 could interact with WNK1, and trigger the ubiquitination and degradation of WNK1 proteins. In addition, low SIAH2 depended on elevated WNK1 proteins to drive HCC malignant features, including proliferation, migration and stemness. Meanwhile, we further found that CBX2 could regulate SIAH2 expressions. CBX2 cooperated with EZH2 to mediate the H3K27me3 enrichment on the promoter region of SIAH2 to suppress its transcriptional levels. High CBX2/EZH2 levels in HCC correlated with poor prognosis of patients. Gene set enrichment analysis (GSEA) further implicated that WNK1 correlates tightly with glycolytic process in HCC samples. WNK1 overexpression was found to notably enhance glycolytic activity, whereas WNK1 deficiency could significantly suppress the HCC glycolysis activity. Lastly, the subcutaneous tumor model further demonstrated that targeting WNK1 was effective to inhibit the in vivo tumor growth of SIAH2low HCC. Collectively, down-regulated SIAH2 expressions induced by CBX2/EZH2 could drive progression and glycolysis via accumulating WNK1 proteins, indicating that CBX2/SIAH2/WNK1 axis is a potential prognostic biomarker and therapeutic vulnerability for human HCC.

RNA fusion transcript panel identifies diverse repertoire of fusions in adult glioma patients with therapeutic implications.

Recurrent gliomas are therapeutically challenging diseases with few treatment options available. One area of potential therapeutic vulnerability is the presence of targetable oncogenic fusion proteins. To better understand the clinical benefit of routinely testing for fusion proteins in adult glioma patients, we performed a retrospective review of 647 adult patients with glioma who underwent surgical resection at our center between August 2017 and May 2021 and whose tumors were analyzed with an in-house fusion transcript panel. Fifty-two patients (8%) were found to harbor a potentially targetable fusion with 11 (21%) of these patients receiving treatment with a fusion-targeted inhibitor. The targetable genes found to be involved in a fusion included FGFR3, MET, EGFR, NTRK1, NTRK2, BRAF, ROS1, and PIK3CA. This analysis demonstrates that routine clinical testing for gene fusions identifies a diverse repertoire of potential therapeutic targets in adult patients with glioma and can offer rational therapeutic options for patients with recurrent disease.

Abstract 6149: STAT3 activation promotes invasiveness and cancer stemness in lung cancer

Abstract Lung cancer is the leading cause of cancer-related death worldwide, featuring a high incidence and low 5-year survival rate. Nearly 40% of patients are diagnosed with metastatic disease when they are first presented to the clinic. However, drug intervention targeting established metastatic tumors remains challenging, a better understanding of therapeutically exploitable mechanisms is urgently needed. This present study aims to explore novel therapeutic intervention against metastatic lung cancer. To understand the tumor-cell-intrinsic pathway that drives metastatic progression, we generated a highly invasive subline of lung cancer cell - A549 (ATCC, USA) by serial selections for Matrigel-coated transwells. The invasive subline A549-i3 shows about 5 times higher invasion potential than the parental cell line A549-Par (p<0.05, Student t-test). Colony-formation Assay and suspension growth in ultra-low attachment plates were used to measure the 2D proliferation ability and anoikis resistance, albeit with no difference. The differential efficacies between A549-Par and A549-i3 against multiple pathway inhibitors were screened using MTT assay in vitro. Notably, A549-i3 is more sensitive towards napabucasin (STAT3 inhibitor) and tipifarnib (FTase inhibitor) when compared with A549-Par, suggesting their potency in targeting cancer stemness and addicted oncogenic driver. Furthermore, napabucasin reduced the migration and invasion abilities of A549-i3 to similar extent as their parent counterpart. Overall, our preliminary data identified STAT3 as a potential therapeutic vulnerability in highly invasive cancer cells. Future work will be focused on identifying pathways responsible to sensitize highly invasive NSCLC cells toward STAT3 inhibition using multiomics approach. This project was supported by the Research Impact Fund (Ref. No.: R4015-19) from the Research Grants Council in Hong Kong. Citation Format: Sai Fung Yeung, Sze Man Chan, Chun Ho Law, Hoi Yin Chan, Kwok Wing Tsui. STAT3 activation promotes invasiveness and cancer stemness in lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6149.

Abstract 4545: Integrative immune-genomic comparison of canonical Ewing sarcoma with Ewing-like mimics identifies potential targets for personalized therapies

Abstract Introduction: Ewing sarcoma (ES) is one of the most common types of small round cell sarcoma (SRCS) from the skeletal origin, characterized by small round blue cell morphology and FET-ETS fusions. However, recent NGS-based technology has dramatically increased the characterization of other SRCS entities that share morphological, pathological, and clinical similarity with canonical ES. Currently, these sarcomas are often treated with similar regimens derived from ES. Whether any subsets of these sarcomas might benefit from personalized anti-cancer strategies remains largely unknown. Methods: To explore potential therapeutic vulnerabilities, 51 FFPE samples from 49 patients (median age 31 years, 65.3% male) diagnosed as ES or its mimics by conventional pathology review were sent to DNA (OncoScreen®Plus) and RNA sequencing (OncoRNA, Burning Rock Biotech) for genomic and immune signature profiling. Tumor samples were classified into 5 groups based on their similarity with classic ES: (a) Canonical ES (n=12), defined as ES with EWSR1-FLI1fusion and a primary bone origin; (b) Non-canonical ES, which were found with either non-EWSR1-FLI1 FET-ETS fusion, or ES in extra-skeletal sites (n=11); (c) Non-FET-ETS fusion SRCS (n=10), defined as SRCS with gene fusions other than FET-ETS family; (d) Fusion-negative SRCS (n=4); and (e) non-SRCS, where the diagnosis was primarily suspected as ES but finally re-diagnosed as another histology (n=12). Results: The NGS-based diagnosis rectified the pathological diagnosis in 9/51 samples, including ES misdiagnosed as other (n=3), other misdiagnosed as ES (n=4), Non-FET-ETS fusion SRCS misdiagnosed as ES (n=1) and ALK fusion sarcoma misdiagnosed as SRCS (n=1). In the remaining 42 samples, 23 diagnoses were confirmed by both NGS and FISH, while 11 SRCS were negative in both NGS and FISH assay, yielding a concordance of 81.0% (34/42). In the 8 samples with discordant diagnosis, a definitive diagnosis was achieved by FISH but not NGS in 1 of 8 (12.5%), and vice versa in 7 of 8 (87.5%). Interestingly, genomic profiling revealed that Non-FET-ETS fusion SRCS had high frequencies of TP53 mutations (40%) and copy number loss (including 30% in CD274 and JAK2; 20% in PTCH1, CDKN2A/B, and MTAP) and featured higher levels of homologous recombination deficiency (HRD) (p=0.024) and chromosome instability (p=0.022) than other groups. Remarkably, non-canonical ES demonstrated upregulated PDCD1 expression (p=0.034). The distribution of immune hot and cold subtypes was comparable among all groups (p=0.334). Conclusion: Our study shows that NGS could efficiently facilitate the definitive diagnosis of ES and its mimics. We also revealed a diverse immune-genomic landscape of these SRCS, indicative of potential therapeutic opportunities targeting HRD and immune check-point in specific subtypes of these sarcoma entities. Citation Format: Zhusheng Zhang, Qiyuan Bao, Junxiang Wen, Zhuochao Liu, Qi Liu, Yuhui Shen, Lin Shao, Bing Li, Song-An Chen, Weibin Zhang. Integrative immune-genomic comparison of canonical Ewing sarcoma with Ewing-like mimics identifies potential targets for personalized therapies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4545.

Abstract 1853: Microparticle-delivered Cxcl9 prolongs Braf inhibitor efficacy in melanoma

Abstract Patients with BRAF-mutant melanoma show significant responses to combined BRAF and MEK inhibition, but most patients relapse within 2 years. A major reservoir for such drug resistance is minimal residual disease (MRD), which is comprised of drug-tolerant tumor cells laying in a dormant state. Towards exploiting potential therapeutic vulnerabilities of MRD, we established a genetically engineered mouse model of BrafV600E-driven melanoma MRD wherein genetic BrafV600E extinction leads to strong but incomplete tumor regression. Transcriptional time-course analysis of tumors after BrafV600E extinction revealed that after an initial surge of immune activation, tumors later became immunologically "cold" after MRD establishment. Computational analysis identified candidate T-cell recruiting chemokines that may be central players in the process, being strongly upregulated initially and then steeply decreasing as the immune response faded. As a result, we hypothesized that sustaining the chemokine signaling could impair MRD maintenance through increased recruitment of effector T-cells. We show that intratumoral administration of recombinant Cxcl9, either naked or loaded in microparticles, significantly impaired the relapse of MRD in BRAF-inhibited tumors, including several complete pathological responses after the triple combination of microparticle-delivered rCxcl9 with BRAF and MEK-inhibition. Our experiments constitute a proof of concept that chemokine-based microparticle delivery systems are a potential strategy to forestall tumor relapse and thus improve the clinical success of frontline treatment methods. Citation Format: Gabriele Romano, Francesca Paradiso, Peng Li, Pooja Shukla3, Lindsay Barger, Olivia El Naggar, John P. Miller, Roger J. Liang, Timothy L. Helms, Jennifer A. Wargo, Francesca Taraballi, James C. Costello, Lawrence N. Kwong. Microparticle-delivered Cxcl9 prolongs Braf inhibitor efficacy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1853.

Abstract 251: Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids

Abstract The promise of precision medicine is based on the idea that genetic alterations present only in tumor cells create vulnerabilities that can be targeted for therapeutic intent. In some instances, somatically mutated driver genes such as KRAS, BRAF or PIK3CA can be targeted with small molecule inhibitors specific for mutant oncoproteins. In other cases therapeutic vulnerability is an indirect consequence, such as response to immune checkpoint inhibitors caused by somatic inactivation of mismatch repair genes and subsequent creation of neo-antigens in tumors. Synthetic lethal vulnerabilities dependent on common somatic alterations would be highly specific therapeutic targets applicable to a large number of cancer patients. We sought to identify potential therapeutic vulnerabilities for colon cancer by performing a genome wide CRISPR knockout screen in a colon cancer organoid in both TP53-Wild-Type and TP53-Knockout backgrounds. We identified 1784 gene knockouts with TP53-dependent effects on Darwinian fitness. Examples include MDM2 and PPM1D knockouts, both of which selectively harmed the TP53 WT organoids. 250 gene knockouts selectively harmed the TP53 KO organoids and represent novel avenues for development of TP53 synthetic lethal targeted therapeutics. We also identified ~1000 gene knockouts under significant negative Darwinian selection in both TP53 WT and TP53 KO organoids which are not known to be common essential genes. Several of these organoid model specific vulnerabilities are in pathways that are downstream of the somatic mutations present in the tumor and are therefore candidates for highly-specific targeted therapeutic intervention. Examples include Werner’s pathway dependencies (WRN, EME1, MUS81) which are known to result from mismatch repair deficiency, WNT pathway dependencies (CTNNB1, PORCN) which result from RNF43 inactivating somatic mutation, and BRAF dependency resulting from its own activating oncogenic somatic mutation. In summary, genome-wide CRISPR knockout library screening of human colon cancer organoids can provide a comprehensive overview of dependencies interacting with either natural or engineered mutations and are a promising novel platform for discovering personalized or pathway based therapeutic targets. Future work will focus on target validation and additional primary screens using our biobank of tumor-normal organoid pairs. Citation Format: Phillip J. Buckhaults, Sana Khalili, Carolyn Banister, Prashanth R. Gokare, Dave Pocalyko, Kurtis Bachman. Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 251.

A TEAD2-Driven Endothelial-Like Program Shapes Basal-Like Differentiation and Metastasis of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDA), with its highly metastatic propensity, is one of the most lethal subtypes of pancreatic cancer. Although recent large-scale transcriptomic studies have demonstrated that heterogeneous gene expressions play an essential role in determining molecular phenotypes of PDA, biological cues for and consequences of distinct transcriptional programs remain unclear. We developed an experimental model that enforces the transition of PDA cells toward a basal-like subtype. We combined epigenome and transcriptome analyses with extensive invitro and invivo evaluations of tumorigenicity to demonstrate the validity of basal-like subtype differentiation in association with endothelial-like enhancer landscapes via TEA domain transcription factor 2 (TEAD2). Finally, we used loss-of-function experiments to investigate the importance of TEAD2 in regulating reprogrammed enhancer landscape and metastasis in basal-like PDA cells. Aggressive characteristics of the basal-like subtype are faithfully recapitulated invitro and invivo, demonstrating the physiological relevance of our model. Further, we showed that basal-like subtype PDA cells acquire a TEAD2-dependent proangiogenic enhancer landscape. Genetic and pharmacologic inhibitions of TEAD2 in basal-like subtype PDA cells impair their proangiogenic phenotypes invitro and cancer progression invivo. Last, we identify CD109 as a critical TEAD2 downstream mediator that maintains constitutively activated JAK-STAT signaling in basal-like PDA cells and tumors. Our findings implicate a TEAD2-CD109-JAK/STAT axis in the basal-like differentiated pancreatic cancer cells and as a potential therapeutic vulnerability.